This method can help answer key questions in the biotechnological and biomedical fields such as what are the molecular basis behind the Warburg and the Crabtree effects. The main advantage of this technique is that it allows the high throughput study of the effects of certain substances in respiratory and fermentative metabolism. Begin by inoculating a 15-milliliter conical tube containing three milliliters of cool, sterile 2%yeast extract peptone dextrose, or YPD broth, with 250 microliters of S.cerevisiae yeast cells preserved in glycerol at negative 20 degrees Celsius.
Incubate the yeast overnight in an orbital shaker at 30 degrees Celsius and 200 rpm. for streaking onto a 60-milliliter, 2%YPD agar-filled Petri dish the next morning. Culture the dish at 30 degrees Celsius until isolated colonies are observed.
Then inoculate a single isolated colony in a new conical tube containing 10 milliliters of cool, sterile 2%YPD broth for overnight culture at 30 degrees Celsius with shaking. For microplate growth-curve setup, add 145 microliters of an appropriate experimental cultural medium to each well of a sterile 10-by-10 well microplate with a lid and inoculate each well with five microliters of the overnight culture. Then incubate the multi-well plate in a microplate reader at 30 degrees Celsius with constant agitation at 200 rpm for 48 hours, measuring the optical density at 600 nanometers every 30 or 60 minutes.
For shaken conical flask growth-curve setup, inoculate 20 milliliter conical flasks containing 4.8 milliliters of an appropriate sterile culture media with 200 microliters of pre-inoculum culture at a 600 nanometer optical density of two for incubation at 30 degrees Celsius and 250 rpm. Agitation at 250 revolutions per minute is critical to achieve a suitable growth in low concentrations of carbon sources that exert respiratory growth as we have observed a reduced yeast growth in respiratory conditions at lower agitation speeds. To allow measurement of the optical density at 600 nanometer every two hours for 24 hours, dilute 100 microliters of the shaken conical flask culture in 900 microliters of distilled water in a one-milliliter spectrophotometer cuvette and gently mix by pipette.
For data processing, in an appropriate statistical package software, create a new project file, open the new table and graph menu, and select XY.Under the sample data menu, select start with an empty data table and a points and connecting line graph. Leave the X section unselected in subcolumns for replicates or error values. In the Y section, select enter 10 replicate values in side-by-side subcolumns and plot mean and error SD.Then click create.
Enter the time at which the OD measurements were obtained in the X column and the OD values obtained from the cultures in the Y column. To identify the exponential growth phase transforming the Y column data into logarithms, click analyze and select the transform analysis. Use Y equals log Y to select the Y values and open the gallery of results.
Select the transform data table, click analyze, and select the linear regression analysis, excluding the optical density values that do not follow a linear trend by selecting the values in the table and depressing control and E.In the data-tables gallery, select the data one table and click analyze. Select the nonlinear regression curve fit analysis and the data sets to analyze, and click OK.Then apply least squares fit to the data, leaving the interpolate section unselected, and click OK.Next, open a new project file, and in the new table and graph menu, select the column choice, start with an empty data table, and the desired graph. Set the graph to plot the mean with the standard deviation, and click create.
Copy the mean or delta time values from the nonlinear fit results table in the gallery of results and paste the data into a column. Finally, click analyze and select the analysis of interest in the column analyses menu to compare the kinetic parameters of the different nutrimental conditions. The growth kinetic threshold values vary between strengths and must be evaluated according to the conditions that we use in the analysis.
Cultures demonstrating a fermentative phenotype have a small lag phase and an exponential phase with a high growth rate. Cultures that obtain energy mainly by oxidative phosphorylation exhibit a longer lag phase with a slow rate of growth during the exponential phase. Calculation of the specific growth rate and doubling time of the growth curves using the exponential growth equation allows for the setting of thresholds for the respiratory and fermentative growth values.
For example, in these representative experiments, the effects of different resveratrol concentrations on S.cerevisiae BY4742 cells reveal the modification of the cell energetic status in response to varying glucose concentrations. Here, supplementation with 10%glucose demonstrates that lower concentrations of ammonium favor fermentative metabolism as evidenced by an extended exponential growth phase in these cultures, while a higher concentration induces a respiro-fermentative metabolism as evidenced by an extended lag phase and a slower growth rate during the exponential phase. Finally, robust differences in fermentative doubling-time values can be observed between these two different yeast strains grown under the same conditions, highlighting the necessity of validating the doubling-time threshold for each strain analyzed.
While attempting this procedure, it's important to remember that this protocol is only used to screen out phenotype. The specific effects on respiration and fermentation must be confirmed by oxygen consumption assays or by the accumulation of fermentation byproducts respectively.
